Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P47989 (xanthine oxidase)
 8,633 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The production of hydrogen peroxide was measured by following the oxidation of dichlorofluorescein (DCFH) entrapped into platelets. Resting platelets produced nanomolar quantities of DCF, which was proportional to the concentration of platelets and was steady during 1 h of incubation. A significant increase of basal DCF fluorescence was induced by stimuli namely thrombin, arachidonic acid, the Ca2+ ionophore A23187 and PMA. The effect of agonists has been also measured in the presence of 3-amino-1,2,4-triazole (AT) or N-ethylmaleimide (NEM), inhibitors of catalase and glutathione peroxidase, respectively. A further significant enhancement of DCF produced in stimulated platelets was detected only in the presence of NEM. A correlation was found between the increase in DCF and externally added hydrogen peroxide or the oxidizing species formed by xanthine oxidase plus acetaldehyde. The yield was not affected by superoxide dismutase and was higher in the presence of AT or NEM. A cooperative effect in the presence of both inhibitors was shown. Glutathione peroxidase plus glutathione diminished the level of DCF to basal levels.
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PMID:Generation of hydrogen peroxide in resting and activated platelets. 162 82

We evaluated the reagents dichlorofluorescin (DCFH) and hydroethidine (HE) for use in flow cytometric analysis of the respiratory burst of alveolar macrophages and monocytes. DCFH and HE are non-fluorescent precursors which can be oxidized intracellularly to the fluorescent compounds dichlorofluorescein and ethidium. Alveolar macrophages (AMs) loaded with either DCFH or HE were analyzed after phorbol myristate acetate (PMA) stimulation. The results, expressed as fmol/cell oxidation product (DCF or ethidium) after fluorometric standardization of the flow cytometer, show that both DCFH (273 +/- 48, mean increase over control +/- SE, fmol/cell, N = 9) and HE (416 +/- 54, N = 11) detected the substantial respiratory burst of hamster AMs. Similar results were obtained with normal human AMs. By using multiparameter analyses, the oxidative response of AMs ingesting opsonized fluorescent latex beads was measured in subpopulations ingesting increasing numbers of particles. A graded increase in oxidation of both DCFH and HE was found in response to increasing phagocytosis. Ingestion of fluoresceinated staphylyococcal bacteria caused similar changes in HE-loaded AMs. Inhibition of respiration with antimycin showed that approximately 95% of the increased oxidative metabolism of hamster AMs ingesting opsonized beads or bacteria was mitochondrial. The remaining 5% (10-40 fmol/cell) is membrane-derived oxidative activity quantitatively similar to that measured in assays of extracellular release of H2O2. Monocytes loaded with either DCFH or HE showed substantial increases in fluorescence after PMA stimulation (mean % increase over control +/- SE at 30 min: 464 +/- 104, DCFH, 505 +/- 156, HE). While DCHF is known to measure H2O2, HE is less well characterized. Exposure of cells to an extracellular source of both superoxide anion (O2-) and H2O2, xanthine oxidase-xanthine, resulted in marked oxidation of intracellular HE. Addition of both superoxide dismutase and catalase blocked this oxidation, indicating that HE can detect both O2- and H2O2. These agents can be useful probes for precise analysis of oxidative metabolism during phagocytosis in AMs and other mononuclear phagocytes.
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PMID:Oxidative metabolism in the alveolar macrophage: analysis by flow cytometry. 231 4

Reactive oxygen species (ROS) play an important role in the pathogenesis of ischemia-reperfusion injury. Extracellular H2O2 generation from bovine pulmonary artery endothelial cells (EC) is known to increase in response to anoxia-reoxygenation (A-R). To determine potential sources of intracellular ROS formation in EC in response to A-R, a fluorometric assay based on the oxidation of 2',7'-dichlorofluorescin was used. Intracellular ROS production declined 40% during 6 h of anoxia (P < 0.05). After A-R, the rates of intracellular ROS formation increased to 148 +/- 9% (P < 0.001) that of normoxic EC (100 +/- 3%). In EC exposed to A-R, allopurinol and NG-methyl-L-arginine (L-NMMA), inhibitors of xanthine oxidase (XO) and nitric oxide synthase (NOS), respectively, reduced intracellular ROS formation by 25 +/- 1% (P < 0.001) and 36 +/- 4% (P < 0.01). Furthermore, at low doses (i.e., 20 microM), deferoxamine and diethylenetriaminepentaacetic acid (DTPA) significantly inhibited intracellular ROS formation. However, at 100 microM, only deferoxamine caused further reduction in DCF fluorescence. In summary, EC respond to A-R by generating increased amounts of XO- and NOS-derived intracellular ROS. The inhibition, to a similar extent, caused by allopurinol and L-NMMA, as well as the effect of deferoxamine and DTPA suggest that the ROS detected is peroxynitrite. Based on these findings and previous work, we conclude that EC generate ROS in response to A-R from at least two different sources: a plasma membrane-bound NADPH oxidase-like enzyme that releases H2O2 extracellularly and XO, which generates intracellular O2-, which in turn may react with nitric oxide to form peroxynitrite.
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PMID:Intracellular generation of reactive oxygen species in endothelial cells exposed to anoxia-reoxygenation. 917 54

Hydroxychavicol (HC; 10 - 50 microM), a betel leaf component, was found to suppress the 2% H(2)O(2)-induced lucigenin chemiluminescence for 53 - 75%. HC (0.02 - 2 microM) was also able to trap superoxide radicals generated by a xanthine/xanthine oxidase system with 38 - 94% of inhibition. Hydroxyl radicals-induced PUC18 plasmid DNA breaks was prevented by HC (1.6 - 16 microM). A 24-h exposure of KB cells to HC (0.5, 1 mM) resulted in 54 - 74% cell death as analysed by a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. HC (10, 50 microM) further suppressed the growth of KB cells (15 and 76%, respectively). Long-term colony formation of KB cells was inhibited by 51% with 10 microM HC. Pretreatment of KB cells with 100 microM HC inhibited the attachment of KB cells to type I collagen and fibronectin by 59 and 29%, respectively. Exposure of KB cells to 0.1 mM HC for 24 h resulted in cell cycle arrest at late S and G2/M phase. Increasing the HC concentration to 0.25 and 0.5 mM led to apoptosis as revealed by detection of sub-G(0)/G(1) peaks with a concomitant decrease in the number of cells residing in late S and G(2)/M phase. Inducing the apoptosis of KB cells by HC was accompanied by marked depletion in reduced form of GSH (>0.2 mM) and the increasing of reactive oxygen species production (>0.1 mM) as analysed by CMF- and DCF-single cell fluorescence flow cytometry. These results indicate that HC exerts antioxidant property at low concentration. HC also inhibits the growth, adhesion and cell cycle progression of KB cells, whereas its induction of KB cell apoptosis (HC>0.1 mM) was accompanied by cellular redox changes.
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PMID:Inducing the cell cycle arrest and apoptosis of oral KB carcinoma cells by hydroxychavicol: roles of glutathione and reactive oxygen species. 1183 9

Thirty-five plant species were selected from the published literature as traditionally used by the Indigenous Peoples of the boreal forest in Canada for three or more symptoms of diabetes or its complications. Antioxidant activities in methanolic extracts support the contribution of these traditional medicines in a lifestyle historically low in the incidence of diabetes. In a DPPH assay of free radical scavenging activity 89% of the methanol extracts had activity significantly greater than common modern dietary components, 14% were statistically equal to ascorbic acid and 23% had activities similar to green tea and a Trolox positive control. Superoxides produced with an NBT/xanthine oxidase assay found scavenging was significantly higher in 29% of the species as compared with the modern dietary components and Trolox. The methanol extracts of Rhus hirta, Quercus alba and Cornus stolonifera performed similarly to green tea's in this assay. Assessment of peroxyl radical scavenging using a DCF/AAPH assay showed 60% of the plant extracts statistically similar to Trolox while R. hirta and Solidago canadensis extracts were greater than green tea, ascorbic acid and Trolox. The majority of the species (63 and 97%, respectively) had scavenging activities similar to ascorbic acid in the superoxide and peroxyl radical scavenging assays.
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PMID:Antioxidant activity in medicinal plants associated with the symptoms of diabetes mellitus used by the indigenous peoples of the North American boreal forest. 1224 96

It is postulated that antioxidant properties of American ginseng root mediate its cardioprotective actions. The antioxidant capabilities of the American ginseng root have been demonstrated previously, however, the berry of the American ginseng has not yet been evaluated. In this study, we tested the American ginseng berry extract (AGBE) for its antioxidant effects in cell-free chemical systems using H(2)O(2)/FeSO(4) to generate hydroxyl radicals which were measured by a fluorescent probe, 2', 7'-dichlorofluorescin diacetate (DCFH/DA). Xanthine/xanthine oxidase was used to generate superoxide anion, which was measured by a fluorescent probe dihydroethidium (DHE). We found that AGBE decreased fluorescence significantly, suggesting that AGBE scavenges oxygen free radicals. We further tested whether AGBE (0.1-1 mg/ml) can protect cardiomyocytes from oxidative injury induced by exogenous or endogenous oxidants. Cells were exposed to either H(2)O(2) or antimycin A (a mitochondrial electron transport chain site III inhibitor that augments mitochondrial oxidant production). The resulting oxidant stress was measured using DCFH/DA and the cell death was assessed using propidium iodide staining. Pretreatment with AGBE (1 mg/ml) significantly attenuated DCF fluorescence by 49% or 85% and reduced cell death by 59% or 63% in cells exposed to H(2)O(2) or antimycin A, respectively. When the effects of extracts from berry and root of American ginseng were compared in cardiomyocytes exposed to antimycin A, we observed that AGBE conferred greater antioxidant protection at the same dose. We conclude that AGBE is a potent antioxidant that protects cardiomyocytes against oxidant-mediated injury and this protection is partly mediated by its free radical scavenging properties.
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PMID:Antioxidant effects of American ginseng berry extract in cardiomyocytes exposed to acute oxidant stress. 1498 Apr 43

Abnormalities in calcium homeostasis and oxidative processes occur in fibroblasts from patients with Alzheimer disease (AD) and in fibroblasts and neurons from transgenic mice bearing a presenilin-1 (PS-1) mutation. Bombesin-releasable endoplasmic reticulum Ca2+ stores (BRCS) are exaggerated in all of these cells. Our previous studies show that H2O2 exaggerates BRCS. The goal of the present study was to determine whether select reactive species exaggerate BRCS in cultured human fibroblasts and to determine if the ability of fibroblasts to handle these specific oxidant species is altered in cells from AD patients. Two fluorescent indicators were used to distinguish different reactive oxygen species (ROS): 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (c-DCF) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM). ROS were produced by a variety of oxidants, including tert-butyl-hydroxyperoxide (t-BHP), hypoxanthine/xanthine oxidase, S-nitroso-N-acetylpenicillamine, 3-morpholinosydnonimine, and sodium nitroprusside. Different oxidants selectively induced various ROS in distinct patterns. These oxidants also induced selective modification in [Ca2+]i and/or BRCS. Of the several oxidants tested, t-BHP was most specific for exaggerating BRCS without affecting basal [Ca2+]i and inducing only c-DCF-detectable ROS. On the other hand, the results show that NO that reacted with DAF-FM was not responsible for alterations in BRCS. Furthermore, the c-DCF-detectable ROS production induced by t-BHP was higher in fibroblasts from AD patients bearing a PS-1 mutation (n = 7) than in those from aged controls (n = 8). The higher production of c-DCF-detectable ROS may underlie the exaggeration of BRCS in fibroblasts from AD patients. Thus, these results are consistent with the hypothesis that abnormalities in selective cellular ROS cause AD-related changes in intracellular calcium regulation.
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PMID:Modification of endoplasmic reticulum Ca2+ stores by select oxidants produces changes reminiscent of those in cells from patients with Alzheimer disease. 1619 25

Previous in vitro studies in our laboratory have shown that mancozeb (MZ) and maneb (MB), both widely used EBDC fungicides, are equipotent neurotoxicants that produce cell loss in mesencephalic dopaminergic and GABAergic cells after an acute 24h exposure. Mitochondrial uncoupling and inhibition were associated with fungicide exposure. Inhibition of mitochondrial respiration is known to increase free radical production. Here the mechanism(s) of neuronal damage associated with MZ exposure was further explored by determining the role that reactive oxygen species (ROS) played in toxicity. Damage to mesencephalic dopamine and GABA cell populations were significantly attenuated when carried out in the presence of ascorbate or SOD, indicative of a free radical-mediated contribution to toxicity. ROS generation monitored by hydrogen peroxide (H(2)O(2)) production using Amplex Red increased in a dose-dependent manner in response to MZ. Inhibition of intracellular catalase with aminotriazole had little effect on H(2)O(2) generation, whereas exogenously added catalase significantly reduced H(2)O(2) production, demonstrating a large extracellular contribution to ROS generation. Conversely, cells preloaded with the ROS indicator dye DCF showed significant MZ-induced ROS production, demonstrating an increase in intracellular ROS. Both the organic backbone of MZ as well as its associated Mn ion, but not Zn ion, were responsible and required for H(2)O(2) generation. The functionally diverse NADPH oxidase inhibitors, diphenylene iodonium chloride, apocynin, and 4-(2-aminoethyl)benzene-sulfonyl fluoride hydrochloride significantly attenuated H(2)O(2) production by MZ. In growth medium lacking cells, MZ produced little H(2)O(2), but enhanced H(2)O(2) generation when added with xanthine plus xanthine oxidase whereas, in cultured cells, allopurinol partially attenuated H(2)O(2) production by MZ. Minocycline, an inhibitor of microglial activation, modestly reduced H(2)O(2) formation in mesencephalic cells. In contrast, neuronal-enriched cultures or cultures treated with MAC-1-SAP to kill microglia, did not show an attenuation of ROS production. These findings demonstrate that Mn-containing EBDC fungicides such as MZ and MB can produce robust ROS generation that likely occurs via redox cycling with extracellular and intracellular oxidases. The findings further show that microglia may contribute to but are not required for ROS production by MZ.
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PMID:Reactive oxygen species generation by the ethylene-bis-dithiocarbamate (EBDC) fungicide mancozeb and its contribution to neuronal toxicity in mesencephalic cells. 1759 14

The epithelial Na(+) channel (ENaC) is a key transporter participating in the fine tuning of Na(+) reabsorption in the nephron. ENaC activity is acutely upregulated by epidermal growth factor (EGF), insulin, and insulin-like growth factor-1 (IGF-1). It was also proposed that reactive oxygen species (ROS) have a stimulatory effect on ENaC. Here we studied whether effects of EGF, insulin, and IGF-1 correlate with ROS production in the mouse cortical collecting duct (mpkCCD(c14)) cells. Western blotting confirmed the expression of the NADPH oxidase complex subunits in these cells. Treatment of mpkCCD(c14) cells with EGF, insulin, or IGF-1 evoked an increase in ROS production as measured by CM-H(2)DCF-DA fluorescence. ROS production caused by a xanthine-xanthine oxidase reaction also resulted in a significant elevation in short-circuit current through the mpkCCD(c14) monolayer. Transepithelial current measurements showed an acute increase of amiloride-sensitive current through the mpkCCD(c14) monolayer in response to EGF, insulin, or IGF-1. Pretreatment with the nonselective NADPH oxidase activity inhibitor apocynin blunted both ROS production and increase in ENaC-mediated current in response to these drugs. To further test whether NADPH oxidase subunits are involved in the effect of EGF, we used a stable M-1 cell line with a knockdown of Rac1, which is one of the key subunits of the NADPH oxidase complex, and measured amiloride-sensitive currents in response to EGF. In contrast to control cells, EGF had no effect in Rac1 knockdown cells. We hypothesize that EGF, insulin, and IGF-1 have a common stimulatory effect on ENaC mediated by ROS production.
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PMID:ROS production as a common mechanism of ENaC regulation by EGF, insulin, and IGF-1. 2313

Apigenin, a natural flavone, is emerging as a promising compound for the treatment of several diseases. One of the hallmarks of apigenin is the generation of intracellular reactive oxygen species (ROS), as judged by the oxidation of reduced dichlorofluorescein derivatives seen in many cell types. This study aimed to reveal some mechanisms by which apigenin can be oxidized and how apigenin-derived radicals affect the oxidation of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (H(2)DCF), a probe usually employed to detect intracellular ROS. Apigenin induced a rapid oxidation of H(2)DCF in two different immortalized cell lines derived from rat and human hepatic stellate cells. However, apigenin did not generate ROS in these cells, as judged by dihydroethidium oxidation and extracellular hydrogen peroxide production. In cell-free experiments we found that oxidation of apigenin leads to the generation of a phenoxyl radical, which directly oxidizes H(2)DCF with catalytic amounts of hydrogen peroxide. The net balance of the reaction was the oxidation of the probe by molecular oxygen due to redox cycling of apigenin. This flavonoid was also able to deplete NADH and glutathione by a similar mechanism. Interestingly, H(2)DCF oxidation was significantly accelerated by apigenin in the presence of horseradish peroxidase and xanthine oxidase, but not with other enzymes showing peroxidase-like activity, such as cytochrome c or catalase. We conclude that in cells treated with apigenin oxidation of reduced dichlorofluorescein derivatives does not measure intracellular ROS and that pro- and antioxidant effects of flavonoids deduced from these experiments are inconclusive and must be confirmed by other techniques.
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PMID:Oxidation pathways underlying the pro-oxidant effects of apigenin. 2611 79


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